Our studies of catalytic proteins will mainly deal with ribulose diphosphate (RuDP) carboxylase, a protein that catalyzes primary carbon dioxide fixation in all living forms. This enzyme also catalyzes the oxygenolytic cleavage of RuDP to form phosphoglycolate and phosphoglycerate, a reaction thought to account for photorespiration which opposes photosynthesis and therefore plant growth. We have developed a one-step purification to isolate this enzyme from two chemolithotrophic and three photolithotrophic bacteria and one green alga and have developed rapid procedures to crystallize the enzyme from several of these sources. We will examine the carboxylase/oxygenase ratios in several of these pure enzymes and probe for glycolate excretion during chemolithotrophic growth, which is inhibited by oxygen. Mechanisms of catalysis by both carboxylase and oxygenase pathways will be compared to test the feasibility of preparing mutants which have higher carboxylase/oxygenase activities of RuDP carboxylase. The subunit primary structures will be compared by tryptic fingerprinting, direct sequence analysis and immunological techniques. Finally, crystals will be grown that will enable elucidation of the crystal structures of the free enzyme and the carboxylated and oxygenated forms. One of the RuDP carboxylases that will be investigated as described is synthesized during autotrophic or mixotrophic growth of the facultative chemolithotroph Thiobacillus intermedius. Under these growth conditions, this organism also synthesizes single-membrane bounded polyhedral inclusion bodies, which seem to contain RuDP carboxylase. These bodies will be isolated and their ultrastructure, enzyme composition, and metabolism investigated in collaboration with Professor Shively. The modification of kinetic properties of RuDP carboxylase by membrane attachment will be examined and the membrane composition determined. The assembly of polyhedral inclusion bodies will be studied in synchronously-dividing T. intermedius. These studies may provide information about the regulation of carbon dioxide assimilation and cell and organelle structure and function.